Control and stabilization of the sample temperature are important criteria for high-resolution NMR experiments, because the measured chemical shift is a physical property that can be sensitive to small temperature changes. NMR experiments employ radio frequency (RF)-pulses that induce sample heating due to dielectric absorption. Differential sample heating among a series of experiments causes difficulties for the analysis of the resulting spectra. For example, for protein structure determination, the side chain protons of proteins are often assigned using TOCSY-type experiments with many RF-pulses and strong sample heating. In contrast, NOESY experiments used to provide distance information feature only a few RF-pulses and cause thus little sample heating. The calculation of protein structures from these experiments depends on the correlation of the chemical shifts in both types of experiments and a high correspondence and control of the sample temperatures in these different experiments can be important in determining accurately protein structures.
Due to these reasons, it is important to control and stabilize the NMR sample temperature with accuracy and precision of the order of 0.1 K. The terms accuracy and precision are used here in their canonical definitions. The accuracy of temperature stabilization refers to the difference between a desired temperature and the actual average sample temperature. Temperature control and stabilization is accurate, if the apparatus creates and maintains the sample temperature at a value desired by the user. The precision of temperature stabilization refers to the standard deviation of the sample temperature. Temperature control and stabilization is precise, if the remaining temperature fluctuations are small, irrespective of whether the temperature is accurate.
In conventional NMR probes, the sample temperature control is achieved with a thermocouple located in the air stream that forms the temperature reservoir for the sample. A measurement of the air stream temperature with accuracy and precision of 0.1 K or below is typical in such conventional setups. However, these external measurements of the air stream do not detect the true sample temperature. RF-induced sample heating, often as large as five degrees K, is not detected and thus not corrected. Thus, despite a precision of 0.1 K, the accuracy of sample temperature stability is 5 K or larger in conventional systems.
An interactive method to correct differential RF-heating in a series of experiments is manual adjustment of the temperature control, by comparing one-dimensional (1D) traces of the spectrum of interest to a reference spectrum. Such a procedure is however error-prone, cumbersome and time-consuming and forbids itself for automated or high-throughput setups. Modern automated setups require that several different experiments on the same sample or the same experiment on different samples are consistently recorded and automatically analyzed. These demands cannot be fulfilled with an interactive method.
Highly accurate and precise measurements of the sample temperature can be achieved in a non-invasive way by using the temperature-dependence of the chemical shift of suitable nuclei as thermometers (e.g. van Geet, A. L., Calibration of methanol nuclear magnetic resonance thermometer at low temperature, Anal. Chem. 42, 679-680 (1970). This is being used in many practical NMR applications, in particular to calibrate the temperature unit of the NMR probe upon installation. However, only if these measurements are carried out in samples with an exactly defined composition, often containing the pure thermometer substance in bulk, the measured chemical shifts can be interpreted as temperatures. Such methods are thus not applicable on samples with a non-standard chemical composition.
A method for using direct NMR measurements to improve the temperature stabilization has been disclosed by H. Keiichiro, Japanese Patent 3-156394 (1991). The disclosed method uses a conventional field-frequency lock unit to extract a second frequency component besides the field-frequency lock signal, calculates the chemical shift difference of these two resonances, and compares the result with values from a reference table for the desired temperature. The outcome of this temperature measurement is then directly used to control the sample heater, either replacing the conventional thermocouple measurements or by adding the two values together. However, since chemical shifts are not only dependent on the temperature, but also on other sample parameters, like the pH value, the salt concentration, etc., the use of this method would require temperature reference tables for each of the infinite number of possible sample compositions. Since these tables are not available, the accuracy achieved by this method is as high as 10 K. Due to this drawback, the temperature controller as described in Japanese Patent 3-156394 is not practicable and has not become widely used.